The present invention relates to models of human diseases and to methods of using these models for identifying compounds effective for the treatment of these diseases. In particular, the present invention represents a new experimental model for the in vivo analysis of human lipoprotein (a) [Lp(a)] and the development of therapeutic strategies to reduce the health risks associated with high levels of this lipoprotein.
Dyslipoproteinaemias are disorders in the metabolism of the lipoproteins which are responsible for transporting lipids such as cholesterol and triglycerides in the blood and the peripheral fluids. Dyslipoproteinaemias consequently are associated with life-threatening diseases linked to hypercholesterolaemia, hypocholesterolaemia or hypertriglyceridaemia, such as atherosclerosis.
Atherosclerosis is a complex, polygenic disease which is defined, on the histological plane, by deposits (lipid or fibro-lipid plaques) of lipids and other blood derivatives in the wall of the large arteries (aorta, coronary arteries and carotid). The plaques, which are more or less calcified according to the progress of the disease, can be associated with lesions and are linked to the accumulation, in the arteries, of fatty deposits which essentially consist of cholesterol esters. The plaques are accompanied by a thickening of the arterial wall together with hypertrophy of the smooth muscle, the appearance of foam cells and the accumulation of fibrous tissue. The plaques are raised on the arterial wall resulting in a stenosis, that is, a narrowing or stricture of the artery. In the worst-affected patients, this stenosis is responsible for the vascular occlusions, such as atheroma, thrombosis and embolisms. Accordingly, excess accumulation of cholesterol (hypercholesterolaemias) can lead to very serious cardiovascular diseases such as infarction, sudden death, cardiac decompensation, cerebrovascular diseases, and the like.
It is particularly important, therefore, to have immediately available treatments which diminish, in some disease situations, the levels of plasma cholesterol or stimulate the efflux of cholesterol (reverse transport of cholesterol) from the peripheral tissues in order to unload the cells which have accumulated the cholesterol in the context of forming an atheroma plaque. Cholesterol is transported in the blood by a variety of lipoproteins including the low density lipoproteins (LDL) and the high density lipoproteins (HDL). The LDLs are synthesized in the liver and are responsible for supplying the peripheral tissues with cholesterol. By contrast, the HDLs pick up cholesterol in the peripheral tissues and transport it to the liver where it is stored and/or broken down.
Numerous studies have correlated elevated plasma levels of lipoprotein (a) [Lp(a)] with increased incidence of cardiovascular disease and stroke (reviewed in Utermann, G., Science (1989) 246, 904-910; Maher, V. M. G., and Brown, B. G., Curr. Opin. Lipidol. (1995) 6, 229-235). Lipoprotein(a) is a complex particle composed of a lipid moiety and two disulfide-linked subunits: apolipoprotein B-100 (apoB-100) and apolipoprotein(a) [apo(a)]. The presence of apo(a), a hydrophilic glycoprotein structurally related to plasminogen, distinguishes Lp(a) from low density lipoprotein (LDL) and confers its characteristic biological and physical properties.
Apolipoprotein B-100 (apoB) is the major protein constituent of very low density lipoproteins (VLDL), low density lipoproteins (LDL) and lipoprotein Lp(a). This protein is the physiological ligand of the LDL receptor, and its plasma concentration is positively correlated with the development of atherosclerosis (Brunzell et al. 1984 Arteriosclerosis 4, 79-93). ApoB-100 is one of the largest known proteins, with a mass of 550 kDa and containing 4536 amino acids (Chen et al. 1986 J. Biol. Chem, 261, 12919-21). This apolipoprotein is only synthesized in the liver. Its plasma concentration is 1.0-1.2 g/l. ApoB-100 plays the major role in transporting cholesterol which is synthesized in the liver through the plasma to the other cells of the organism. Another version of apoB, i.e. apoB-48, is present in the chylomicrons. In humans, apoB-48 is synthesized in the intestine. ApoB-48 has a mass of 260 kDa and contains 2152 amino acids which linearly correspond to 48% of the N-terminal end of apoB-100 (Powell et al., 1987, Cell 50, 831-40). Since the C-terminal moiety of apoB-100 contains the zone for binding the apoB-100 to the LDL receptor, apoB-48 does not attach to this latter receptor and behaves in a different manner metabolically.
To study the disease states relating to dysliproproteinaemias, it is advantageous to have available an animal model which expresses a protein or a protein complex which is associated with a risk for a disease which is linked to dyslipoproteinaemias. Such an animal model would be particularly advantageous for understanding these diseases and, more specifically, the regulatory mechanisms which these proteins or protein complexes initiate. This would make it possible to test, rapidly and in vivo, a considerable number of therapeutic agents or compounds for the purpose of detecting a potential activity associated with the expression of the proteins. Furthermore, such a model would be of interest for developing novel therapeutic methods for treating these types of diseases, such as methods which are based on gene therapy. The in vivo analysis of Lp(a), an independent atherosclerosis risk factor in humans, has been limited in part by its restricted distribution among mammals. Apo(a) is naturally present exclusively in old world monkeys, humans, and one non-primate species, the European hedgehog. Such limited distribution of apo(a) among mammals has limited studies of its in vivo properties.
Accordingly, the present invention relates to animal models of disease states involving Lp(a), including atherosclerosis, to enable screening and identification of compounds for the treatment of these diseases, in particular, atherosclerosis.
Generally speaking, the murines, namely mice, rats and guinea pigs, are the most widely used animal models. They are easy to manipulate and inexpensive. Unfortunately, these small mammals are not always compatible with the intended application because they are not always representative of humans and their metabolism. Chimpanzees are used for testing therapeutic agents and vaccines directed against various diseases, including AIDS and cancer. However, the very substantial cost incurred in using chimpanzees as a model system constitutes a major and compelling handicap with regard to its use.
Despite the drawbacks of a system using mice, the development of transgenic mice expressing human apo(a) cDNA provided a means to test hypotheses accounting for the effect of Lp(a) on the vasculature. Specifically, these mice have been used to examine the ability of apo(a) to promote atherogenesis by inhibition of plasmin formation and associated consequences (Lawn, et al., Nature (1992) 360, 670-672; Grainger, et al., Nature (1994) 370, 460-462). Studies of apo(a) transgenic mice have also led to several important insights into Lp(a) assembly, including the observation that apo(a) was unable to form a covalent association with LDL containing murine apoB (Chiesa, et al., J Biol Chem (1992) 267, 24369-24374). This result, coupled with evidence for Lp(a) formation when the mice were infused with human LDL or expressed a human apoB transgene (Linton, et al., J Clin Invest (1993) 92, 3029-3037; Callow, et al., Proc Nat""l Acad Sci, USA (1994) 91, 2130-2136) suggested that murine apoB lacked structural requirements necessary for Lp(a) assembly. This was not a completely unexpected finding in light of the absence of the apo(a) gene in mice and the sequence specific interactions between apo(a) and apoB believed to mediate Lp(a) assembly in humans. Two studies using site specific mutagenesis of human apoB transgenes in mice have reported localization of a single cysteine in human apoB (Cys 4326) which provides the site of attachment for apo(a) (Callow, M. J., and Rubin, E. M., J Biol Chem (1995) 270, 23914-23917; McCormick, et al., (1995) 92, 10147-10151).
Most recently, the regulation of apo(a) gene expression has been studied in transgenic mice containing a human apo(a) genomic clone (Frazer, et al., Nat Gen (1995) 9, 424-431). This transgene comprised the apo(a) gene along with its native promoter and cis acting elements present within the approximately 60 kb of 5xe2x80x2 and 80 kb of 3xe2x80x2 flanking DNA. The transgene was more efficiently expressed (i.e. all the apo[a] transgenic founder lines created containing an intact transgene expressed apo[a]) and resulted in significantly higher plasma apo(a) levels than observed in mice containing an apo(a) cDNA construct. A surprising finding in mice expressing the human apo(a) genomic transgene was the profound sex hormone-induced changes in apo(a) expression, far surpassing the magnitude of androgen and estrogen related changes observed in humans. These changes were, however, qualitatively similar in humans and transgenic mice. In both cases, these hormones lower apo(a) plasma levels.
The use of genomic clones in creating transgenic animals generally has several advantages over the use of cDNA constructs. Most importantly, transgene expression is regulated in an appropriate manner and is independent of its site of integration. This approach has been limited to the production of transgenic mice for large genes such as apo(a), however, due to the technical difficulty of manipulating extremely large genomic clones and the lower efficiency of transgenesis in other animals.
Although transgenic mice have been created containing Lp(a), the small size of the mouse and the differences in lipoprotein profiles between mice and humans has precluded various studies. Accordingly, it is desirable to identify an animal model system which is closer to man but avoids the expense associated with primate model systems.
Thus, there is a need in the art for an appropriate animal model of Lp(a)-mediated diseases and disorders. There is a further need in the art for a transgenic animal, that expresses human Lp(a). Moreover, to be truly useful, a transgenic animal must express the constituent Lp(a) proteins at a high enough level, and must permit covalent association of the constituents so as to produce physiologically relevant levels of Lp(a). To date, the technical difficulties in generating such an animal have not been overcome, until the present invention.
The present invention overcomes these deficiencies of the prior art, by providing a useful transgenic animal that produces physiological levels of human Lp(a), despite the difficulties in achieving such an invention.
In particular, the invention relates to transgenic animals expressing both the human apolipoprotein(a) [apo(a)] and apolipoprotein B (apoB) genes. These animals express human lipoprotein(a) [Lp(a)], the complex particle composed of a lipid moiety and two disulfide-linked subunits of apo(a) and apoB.
In accordance with the present invention, there is provided a transgenic rabbit which has in its genomic DNA sequences that encode apolipoprotein (a) and apolipoprotein B polypeptides which are capable of combining to produce lipoprotein (a).
The present invention also provides a transgenic rabbit capable of producing lipoprotein (a) wherein the rabbit develops human-like atherosclerotic lesions when fed a cholesterol rich diet.
The present invention also provides for transgenic rabbits capable of producing lipoprotein (a) having exogenous human DNA sequences. The DNA sequences may be selected from genomic DNAs and cDNAs.
As stated above, the transgenic rabbits of the present invention have in their genomic DNA sequences that encode apolipoprotein (a) and apolipoprotein B. The present invention provides transgenic rabbits capable of producing lipoprotein (a) having liver cells which express the DNA sequences and transgenic rabbits capable of producing lipoprotein (a) having testes cells which express the DNA sequences.
Yet another aspect of the present invention is a transgenic rabbit having a stable plasma level of apolipoprotein (a) polypeptide throughout its sexual maturity.
The present invention also provides a process for creating a transgenic rabbit which is capable of expressing apolipoprotein (a) and apolipoprotein B polypeptides and which comprises combining the germ line cells of a first rabbit which is capable of expressing apolipoprotein (a) with the germ cells of a second rabbit which is capable of expressing apolipoprotein B. Germ line cells include, for example, eggs and sperm.
Yet another aspect of the present invention is the provision of a process for creating a transgenic rabbit which is capable of expressing apolipoprotein (a) and apolipoprotein B polypeptides and which comprises mating a first rabbit which is capable of expressing apolipoprotein (a) with another rabbit which is capable of expressing apolipoprotein B.
In a preferred method for creating a transgenic rabbit capable of expressing lipoprotein (a), the rabbit expressing apolipoprotein B protein is provided by injecting a rabbit embryo with a phagemid which contains the human apolipoprotein B gene.
In a preferred method for creating a transgenic rabbit capable of expressing lipoprotein (a), the rabbit expressing apolipoprotein (a) is provided by injecting a rabbit embryo with a yeast artificial chromosome which contains a human apolipoprotein (a) gene.
In yet another method for creating a transgenic rabbit capable of expressing lipoprotein (a), the rabbit expressing apolipoprotein (a) is provided by introducing into a rabbit embryonal stem cell at least one human DNA fragment encoding apolipoprotein (a) protein, combining the stem cell with a rabbit blastocyst and transferring the embryo to a recipient female rabbit.
In yet another method for creating a transgenic rabbit capable of expressing lipoprotein (a), the rabbit expressing apolipoprotein B is provided by introducing into a rabbit embryonal stem cell at least one human DNA fragment encoding apolipoprotein B protein, combining the stem cell with a rabbit blastocyst and transferring the embryo to a recipient female rabbit.
In still yet another method for creating a transgenic rabbit capable of expressing lipoprotein (a), the rabbit carrying and expressing apolipoprotein (a) is provided by infecting a rabbit blastomere with a retrovirus comprising at least one human DNA fragment encoding apolipoprotein (a) protein and transferring the blastomere to a recipient female rabbit.
In yet another method for producing a transgenic rabbit capable of expressing lipoprotein (a), the rabbit carrying and expressing apolipoprotein B is provided by infecting a rabbit blastomere with a retrovirus comprising at least one human DNA fragment encoding apolipoprotein B protein, and transferring the blastomere to a recipient female rabbit.
The present invention also provides a method for determining whether a compound can inhibit a disease or disorder linked to lipoprotein (a) expression or metabolism comprising comparing a first transgenic rabbit which is capable of producing human lipoprotein (a) and manifests a disease or disorder linked to human lipoprotein (a) and which has been treated with the compound, with a second transgenic rabbit which is capable of producing human lipoprotein (a) and manifests a disease or disorder linked to lipoprotein (a) and which has not been treated with the compound. The present invention also provides for utilizing this method wherein the disease or disorder is selected from the group consisting of atherosclerosis, cardiovascular disease, ischemia, stroke, restenosis, coronary artery disease, peripheral occlusive arterial disease, myocardial infarction, thrombosis, and an undesirable lipid profile.
The present invention also provides a method for determining whether a compound can inhibit assembly of a lipoprotein (a) particle comprising comparing the level of lipoprotein (a) production in a first transgenic rabbit which has been treated with said compound and which is capable of producing human lipoprotein (a) to the level of production of lipoprotein (a) in a second transgenic rabbit that has not been treated with said compound and which is capable of producing human lipoprotein (a). The present invention also provides for utilizing this method wherein the compound is selected from the group consisting of antisense nucleic acids, and intracellular binding proteins.
The present invention provides transgenic rabbits, an appropriate animal model of lipoprotein (a)-mediated diseases and disorders. The rabbits express the constituent lipoprotein (a) proteins at a high level so as to facilitate covalent association of the constituents to produce physiologically relevant levels of lipoprotein (a). The rabbits offer significant advantages over transgenic mice, due, in part, to the larger size of the rabbit which facilitates study of vascular injury and restenosis. Furthermore, although rabbits are similar to mice in lacking apolipoprotein (a) and lipoprotein (a), the rabbit""s lipoprotein profile more closely mimics that of humans with LDL as the predominant plasma protein. In addition, rabbits also develop well-characterized human-like atherosclerotic lesions when fed a cholesterol-rich diet.